Significance: The TopoisomeraseII (TopII) family is an important class of topoisomerases that produces transient 52 DNA-protein crosslinks. Drugs targeting these enzymes act by stabilizing that crosslink and producing a covalent DNA-protein complex. Despite their clinical efficacy, drugs that target TopII (TopII poisons) may lead to negative side-effects, including secondary malignancies. The repair of TopII-DNA complexes is poorly understood. Recently, a human 52-tyrosine phosphodiesterase (hTDP2) has been identified for the excision of TopII-DNA adducts. As hTDP2 repairs the cytotoxic adducts produced by TopII poisons, it is likely that effective inhibition of hTDP2 can be targeted to improve the efficacy of TopII poisons and reduce the side-effects. It has been shown that the knockdown/knockout of TDP2 activity in A549 (non- small cell lung cancer cell line, NSCLC) and DT40 cells increased sensitivity to the TopII- targeting agent etoposide, demonstrating that hTDP2 can be a target for adjuvant chemotherapy. Our hypothesis is effective inhibition of hTDP2 will increase the cancer cell killing efficacy of TopII poisons like etoposide. Therefore, hTDP2 inhibitor screening, especially in a high-throughput (HT) manner, will expedite drug discovery. Innovation and Novelty: We have developed and statistically validated a novel assay using p- nitrophenyl-thymidine-52-phosphate (T5PNP) as a substrate. This assay can be used in continuous or endpoint colorimetric assays in a multiwell format. Specific Aim: Aim 1. Identification of Inhibitor. Aim 2. Development and characterization of inhibition. Expected Results: We will discover few (2-3) chemical probes, that are acceptable potent (IC50<1 mM and if possible preferably < 100 nM) and selective (counter screened against human 32-tyrosyl DNA phosphodiesterase, hTDP1 with at least 10x specificity) inhibitors of hTDP2 that will increase etoposide mediated A549 cell killing. Future Plan and long term goal: We will test the small molecule inhibitors as an adjuvant to etoposide or doxorubicin treatment in different cancer cell lines and orthotopic model of lung cancer.